An in vitro model of human gastrointestinal digestion was introduced to investigate the effects of surface charge of cellulose nanoparticles on emulsion structure during gastric phase, lipase activity, bile salt diffusion, and free fatty acid (FFA) release. Four carboxymethylated cellulose nanofibrils (CNF; C0, C0.36, C0.72, and C1.24) were used, showing different surface charge (p < 0.05). First, four carboxymethylated CNFs had no inhibition effects on lipase activity and bile salt diffusion. Moreover, we found that the lipid emulsion containing CNF formed gel structure to induce oil droplets aggregation during simulated gastric phase. Additionally, the particle surface charge greatly influenced the gel structure of emulsion where a denser gel structure was observed in the C0 (lowest surface charged CNF) stabilized emulsion. Finally, the released FFA results showed that the formed gel structure lowered the lipid emulsion digestion attributed to the restricted adherent area of oil droplets for lipase and bile salt.